The major objective of this project is to develop hematopoietic stem cell (HSC) gene therapy for patients with Fanconi anemia (FA). FA is an autosomal recessive syndrome characterized by congenital abnormalities, predisposition to malignancy, and bone marrow failure, the latter being the major cause of morbidity and mortality. Allogeneic HSC transplantation from unaffected donors is the only proven curative treatment for patients suffering from marrow failure. However, transplantation for patients with unrelated donors has been associated with significant toxicity, and outcome for these patients has been less successful than for patients with an HLA-matched sibling donor. In addition, recent data suggest that graft- versus-host disease (GVHD) increases the incidence of head and neck cancers, and leads to increased mortality in patients with FA. Gene replacement therapy using autologous hematopoietic stem cells is a potential alternative treatment modality, particularly since gene-corrected FA cells have a survival advantage. In addition, FA cells are highly sensitive to low doses of cyclophosphamide, which could be used to increase the proportion of genetically modified cells and also eliminate unmodified cells. Gene therapy for FA, however, has to date been limited by low gene transfer efficiency resulting in only transient detection of genetically modified cells and, ultimately, no clinical benefit. Part of this problem has been the limitation of gammaretroviral vectors, which require cell division and extended cell culture periods for efficient transduction. This is a particular problem for FA, since FA cells have an increased rate of apoptosis, and thus, their ability to divide and grow in culture is significantly reduced. In contrast to gammaretroviral vector, lentiviral vectors do not require cell division for transduction and can transduce stem cells even with very short transduction protocols. Using mouse and large animal models, we have recently demonstrated efficient lentiviral HSC gene transfer with an overnight transduction protocol. Thus, we propose to 1) complete preclinical studies of FANCA and FANCC lentiviral vectors in human FA cells and in FANCA-/- and FABCC-/- mice and optimize procedures for clinical transduction, 2) determine safety and feasibility of infusing gene-corrected cells, 3) determine persistence and in vivo growth advantage of gene-corrected cells, 4) evaluate whether cyclophosphamide can enhance the survival advantage of gene- corrected FA cells, and 5) analyze lentiviral integration sites and monitor for the development of dominant clones in patients after infusion of gene-modified cells. Our ultimate goal is to improve or cure the hematopoietic symptoms in FA patients by lentivirus-mediated HSC gene transfer, and develop methodologies that would be applicable to other genetic disorders and hematological diseases.